Vaccines have played, and still play, a major role in reducing the impact of infectious diseases on global health. The first generation of vaccines was based on attenuated or inactivated pathogens. These full-pathogen-based vaccines have proven extremely effective and, in some cases, have (e.g. small pox) led to the complete eradication of the target pathogen. There are however serious concerns associated with using full-pathogens for immunization as these have been seen to induce severe side effects at some frequency in populations, underscoring the need to develop safer vaccines (Plotkin S A et. al 2005). Along with the recent advances in recombinant DNA technology and genetic engineering, modern vaccine research has put effort into identifying critical antigenic targets of neutralizing antibodies with the aim of developing so called ‘subunit vaccines’ composed solely of well-defined, purified antigen components (Murray K. et al. 1988). The immunogenicity of subunit vaccines based on low valency soluble protein is, unfortunately, low compared to that of full pathogen-based vaccines. To induce a high-titer antibody response it is thus often necessary to use high antigen doses, booster administrations, and co-administration of adjuvants and even so these subunit vaccines are generally not capable of inducing long-term protective immunity. This is indeed exemplified by the many vaccine failures observed with low valency soluble proteins during the past several years and have led to the conjecture that the size, valency, and the spatial assembly of the vaccine antigen component are critical parameters for optimal activation of the immune system. Virus-like particles (VLPs), which are both highly immunogenic and safe, represent a major advancement in the development of subunit vaccines, combining many of the advantages of full pathogen-based vaccines and simple recombinant subunit vaccines. VLPs are composed of one or several recombinantly expressed viral proteins which spontaneously assemble into macromolecular particulate structures mimicking the morphology of the native virus coat—but lacking infectious genetic material. The particulate nature and size of VLPs (22-150 nm) appears to be optimal for efficient uptake by professional antigen presenting cells, particularly dendritic cells (DCs) as well as for entry into lymph vessels and hence VLPs efficiently stimulate both the humoral and cellular arms of the immune system (Bachmann, M F, Jennings, G T. 2010). Furthermore, surface structures presenting an antigen at high density, with regular spacing, and with consistent orientation are characteristic of microbial surface antigens for which the mammalian immune system has evolved to respond vigorously to. At the molecular level, the presentation of an epitope at high density, while being regularly spaced, and with consistent orientation enables efficient cross-linking of B-cell receptors (Bachmann, M F and Zinkemagel, R M. 1997) leading to strong B-cell responses, even in the absence of T-cell help (Bachmann, M F et al., 1993; Chackerian et al., 1999; Kouskoff, V. et al., 2000) and cumulative data from several studies indicate that B-cells, in fact, discriminate antigen patterns via the degree of surface Ig-cross-linking and use antigen repetitiveness as a self/nonself discriminator.
It has long been an attractive goal to exploit the VLPs as an immunogenicity-boosting platform for inducing immune responses against heterologous antigens by using them as molecular scaffolds for antigen presentation. Antibodies are believed to be the primary effectors of all current prophylactic microbial vaccines and hence the main focus for developing VLP-based vaccines is to induce strong humoral responses, which is especially true when targeting self-antigens. Traditionally this has been achieved either by incorporation of antigenic epitopes into VLPs by genetic fusion (chimeric VLPs) or by conjugating antigens to preassembled VLPs. The chimeric VLP approach is to date the most common method for displaying heterologous epitopes on VLPs (Pumpens, P and Grens, E. 2001; Bachmann, M F and Jennings, G T, 2004a; Chackerian, 2007; Grgacic, E V L. and Anderson, D A. 2006). However, this strategy is severely limited by both the size and nature of epitopes that can be inserted into VLPs, especially in their immunodominant regions, and it has in general not been possible to insert peptides longer than 20 amino acids without disrupting the fragile self-assembly process of the VLPs. In addition, this approach requires that critical epitopes have already been identified in the target antigen and that they can be presented in an immunodominant region on the VLP surface while maintaining their native conformation. Therefore, despite a still growing understanding of the VLP structure/assembly process, generating chimeric VLPs is still a trial-and-error process and it remains impossible to predict whether individual peptides will be compatible with VLP assembly or whether insertions will be immunogenic. Finally, due to the small size of inserted peptide sequences the induced antibody response will functionally be essentially monoclonal, which in some cases will set a limit to the potency of protection. On the other hand, chemical conjugation, e.g. through chemical biotinylation of exposed lysine residues, allows the attachment of diverse kinds of target antigens (incl. non-protein targets) to VLPs and this approach is, in principle, not restricted by the size of the antigen (Raja K S. et al. 2003). However, so far only shorter peptides have successfully been coupled at high density and with consistent orientation to the surface of VLPs (Bachmann M F, Jennings G T. 2011) and in the case of larger antigens it remains highly challenging to control both the orientation and the total amount/stoichiometry of the coupled antigen, affecting both the density and regularity of displayed epitopes, and thus potentially limiting the immune response. In addition to this, chemical coupling procedures are rarely compatible with large scale vaccine production. As a result the current technologies are not sufficient to ensure VLP display of antigens at high density, with regular spacing, and with consistent orientation, which are three critical factors for obtaining strong and long lasting activation of the immune system.
In brief:                Induction of a strong and long lasting immune response to pathogens as well as disease associated antigens is very difficult to obtain with simple subunit vaccines.        Virus-like particle (VLP) presentation of antigens has proven to be very efficient in inducing the highly functional long-term immune responses.        Coupling of an antigen onto the surface of a VLP, at high density, and with a consistent orientation for optimal epitope display, poses a major biotechnological challenge.        